Fast release of neurotransmitters at CNS synapses is vital for brain function. At typical synapses, action potential depolarization of the presynaptic terminal activates a set of voltage gated Ca2+ channels, the N-, P/Q- and possibly R-type channels (known collectively as CaV2 channels) which generate a brief but large rise in free Ca2+ near the Ca2+ sensor molecule synaptotagmin. This unleashes vesicle fusion and transmitter release. We seek to understand the cell biology that puts the Ca2+ channels within the presynaptic active zone in appropriate abundance, stoichiometry and proximity to the release machinery. Our published experiments suggest that the proper deployment of presynaptic Ca2+ channels depends on the operation of 'slots' that interact with channel proteins to control their participation in neurotransmission. The evidence rests on the overexpression of mutant channels engineered to be Ca2+-impermeable. These channels strikingly reduce the ability of wild-type channels to support transmission, as if channels competed for a limited number of binding sites. One class of slots prefers P/Q-type channels. Another class accepts N-type but not P/Q-type channels. The main goal of our proposal is to clarify the nature of these putative CaV2-binding sites. We will delineate their physiological impact, their molecular and cellular basis, and their effect on the spatial organization of CaV2 channels. To achieve these goals, we will capitalize on recent experimental advances in our lab. First, we devised an efficient optical method to measure synaptic vesicle fusion and to dissect the contributions of each CaV2 channel to presynaptic Ca2+ influx. We engineered new red fluorescent protein reporter of exocytosis to be able to monitor simultaneously vesicle fusion and Ca2+ entry through CaV2 channels, using a green Ca2+ reporter, to see how well each CaV2 is coupled to vesicle fusion. Second, we will combine optical tests of synaptic activity with immunocytochemistry to find out if the apparent cap on the number of CaV2 channels mediating vesicle fusion is due to trafficking restrictions, a limited number of synaptic anchoring sites or both. Third, we devised a new approach to test whether potential synaptic anchor proteins really do anchor Ca2+ channels close to the machinery of vesicle fusion. Finally, we will dissect the organization of synaptic landmarks, CaV2 channels and synaptic organizing proteins with the 80-90 nm resolution of STED microscopy. Each CaV2 has a unique sensitivity to membrane potential and neuromodulating hormones and roles in migraine, neuropathic pain and epilepsy. Thus probing channel-slot interactions will help us to understand the regulation of neurotransmission by synaptic activity and neuromodulators in health and diseases.